1. Field of the Invention
The present invention generally relates to methods of drug screening and treating HIV, and more particularly, to inhibiting the binding of the HIV gag protein with the plasma membrane of an infected cell thereby reducing replication of the HIV virus.
2. Related Art
Human immunodeficiency virus (HIV) infection causes the acquired immunodeficiency syndrome (commonly known as AIDS). HIV is a retrovirus that primarily infects T cells expressing the CD4 glycoprotein, i.e., CD4+ T-cells, which are also known as helper T-cells. HIV virus multiplies in helper T-cells and quickly destroys the host helper T-cells, resulting in cellular immunity depression and leaving the infected patient susceptible to opportunistic infections, malignancies and various other pathological conditions. Ultimately, HIV infection can cause depletion of helper T-cells and collapse of a patient's immune defenses. Not surprisingly, HIV-infected individuals and AIDS patients typically develop AIDS-related conditions such as AIDS-related complex (ARC), progressive generalized lymphadenopathy (PGL), dementia, tropical paraparesis, Kaposi's sarcoma, thrombocytopenia purpurea, herpes infection, cytomegalovirus infection, Epstein-Barr virus related lymphomas among others.
HIV is a nontransforming human retrovirus belonging to the lentivirus family. Two genetically different but related forms of HIV, called HIV-1 and HIV-2, have been isolated from patients with AIDS. HIV-1 is the most common type associated with AIDS in the United States, Europe, and Central Africa, whereas HIV-2 causes similar disease principally in West Africa.
Retroviral genomes encode a polyprotein called Gag that contains all of the viral elements required for virus assembly (1). Subsequent to ribosomal synthesis, the Gag proteins are directed to punctuate sites on plasma and/or endosomal membranes, where they assemble and bud to form immature virions (2-4). Approximately 1,500-5,000 copies of Gag contribute to each virus particle (4, 5). During or shortly after budding, the Gag proteins are cleaved by the viral protease into the matrix (MA), capsid (CA), and nucleocapsid proteins, which rearrange to form mature and infectious virions.
Membrane binding is mediated by Gag's N-terminal MA domain and, for most retroviruses, depends on posttranscriptional N-terminal acylation. The MA domain of HIV [types HIV-1 and 2] requires an N-terminal myristyl group that functions in concert with a group of conserved basic residues to promote membrane selection and binding. Mutations that either block myristoylation (6-10) or disrupt the basic patch (11-14) inhibit membrane binding in vitro and can lead to aberrant targeting of Gag to the cytoplasm and/or intracellular membranes in vivo. Intact Gag binds more tightly to membranes than the isolated MA protein, which led to the suggestion that binding may be mediated by a myristyl switch mechanism (9, 15-18). Consistent with this hypothesis, NMR-based structural studies confirmed that the myristyl group of MA can adopt myristate sequestered [myr(s)] and exposed [myr(e)] conformations, and that protein self association promotes myristate exposure (19).
The ability of HIV-1 Gag to colocalize at specific subcellular membranes is essential for viral replication and pathogenesis and may be important for establishing intracellular viral reservoirs that are protected from the immune system (13, 14, 20-25). In most hematopoietic cells, Gag molecules assemble and bud from the plasma membrane (PM), possibly by indirect routing by endosome/multivesicular body (MVB) compartments (14, 22). Gag may also be transiently routed through the nucleus before assembly (26-28). In primary macrophages, budding occurs mainly in MVBs (22-25). Recent studies indicate that the ultimate localization of Gag at virus assembly sites depends on phosphatidylinositol (PI)4,5-bisphosphate [PI(4,5)P2 (21), a member of a family of differentially phosphorylated phosphatidylinositides that serve as membrane markers for specific cellular proteins (29-31). PI(4,5)P2 is normally associated with the inner leaflet of the PM (30). Depletion of PI(4,5)P2 inhibits virus assembly and leads to accumulation of Gag at the membranes of late endosomes and MVBs. Induction of PI(4,5)P2-enriched endosomes also retargets Gag to endosome/MVBs and induces intravesicle budding. In both cases, virus production is severely attenuated (21). Substitution of MA by the membrane-binding N terminus of Fyn kinase reduces the sensitivity of virus assembly to PI(4,5)P2 manipulation, suggesting that PI(4,5)P2-dependent membrane selection is mediated by the MA domain of Gag (21). Consistent with this hypothesis it has been recently shown that phosphoinositides are capable of binding to the MA domain of unmyristoylated Gag fragments and promoting their assembly in vitro into virus-like particles (32, 33).
There has been a great deal of effort in developing methods and pharmaceutical compounds for treating HIV infection and AIDS. The therapeutic approaches have been mostly focused on a limited number of drug targets, namely HIV reverse transcriptase, HIV protease, and HIV integrase. However, HIV typically undergoes active mutations as it multiplies and renders the virus resistant to the inhibitors administered to patients. Combination therapy, generally referred to as HAART (highly active anti-retroviral therapy), has been developed in which a combination of different anti-HIV inhibitors is administered to a patient. However, viral resistance to combination therapies still frequently develops.
Therefore, although limited success for controlling HIV infection and AIDS has been achieved with previously developed anti-HIV compounds, there is a need for alternative therapeutic approaches that overcome the shortcomings of currently available drugs.